1. Field of the Invention
The present invention relates to methods of stimulating phagocytic activity and compositions therefor.
2. The Prior Art
A considerable amount of work has heretofore been done in connection with the natural phenomenon known as phagocytosis, in terms of attempting to understand the mechanism as well as attempting to enhance, or stimulate the process. It is, for example, known that hyaluronic acid (HA) and particularly the sodium salt thereof (Na-HA) both inhibits and stimulates phagocytosis, depending on conditions. Thus, Forrester and Balazs (1979) in Inhibition of phagocytosis by high molecular weight hyaluronate. (Submitted for, but not yet published); and Sebag, Balazs, Eakins, Bhattacherjee, and Kulkarni, (1979), in The effect of hyaluronic acid on prostaglandin synthesis and phagocytosis by mononuclear phagocytes in vitro, (Submitted for publication to The Journal of Cell Biology.) have shown that sodium hyaluronate (Na--HA) of various molecular sizes (480-2300.times.10.sup.3) inhibits the phagocytic activity of peritoneal macrophages. This inhibition depends on the viscosity of the solution to which the cells are exposed. Complete inhibition of phagocytosis of plastic beads was found to occur when the medium had a specific viscosity of 8 to 9. (Balazs, The effects of viscosity on phagocytosis [unpublished data].) This inhibition was not specific to Na-HA because gelatin and DNA solutions of approximately the same viscosity also caused complete inhibition. Sulfated glycosaminoglycans, however, in similar and even somewhat higher concentrations, and the oligosaccharides of HA did not cause inhibition, nor did they show interference with the inhibition of the higher molecular weight Na-HA.
Brandt, in The effect of synovial hyaluronate on the ingestion of monosodium urate crystals by leucocytes. Clinica Chimica Acta 55, 307-315 (1974), showed that various molecular weight Na-HA preparations (2.94-0.86.times.10.sup.6) inhibit the uptake of urate crystals by blood leucocytes. Others have shown that the phagocytosis of staphylococci is diminished in the presence of synovial fluid [Bodel and Hollingworth, Comparative morphology, respiration, and phagocytic function of leukocytes from blood and joint fluid in rheumatoid arthritis. J. of Clin. Invest. 45(4), 580-589 (1966)]. The phagocytosis of encapsulated Group A streptococci by human blood leucocytes was also studied in vitro by Hirsch and Church [Studies of phagocytosis of Group A Streptococci by polymorphonuclear leucocytes in vitro. J. Exp. Med. 111, 309-322 (1960)]. These workers demonstrated that the Na-HA of the capsule has antiphagocytic activity. They also determined that human, but not rabbit, serum, contains a factor which counteracts the antiphagocytic effect of the capsular Na-HA. The nature of this factor was not elucidated by them but an antibody to Na-HA or a Na--HA depolymerizing enzyme was excluded.
The stimulatory effect of Na-HA on the phagocytic activity of both mononuclear and polymorphonuclear phagocytes was recently demonstrated. Thus, Hakansson, Halgren, and Venge [Hyaluronic Acid--A New Opsonin. (Personal communication; 1978)] showed that Na-HA with a molecular weight higher than 0.7-2.times.10.sup.6 in low concentration (1-10 .mu.g/ml) stimulates the initial rate of phagocytosis of plastic beads by human blood leucocytes. This stimulatory effect is specific for serum opsonized particles. The uptake of IgG coated particles was not stimulated.
Forrester & Balazs, supra, found that phagocytosis of peritoneal machrophages is also stimulated by Na-HA at low concentrations. The mechanism of both the stimulation and inhibition for mono- and polymorphonuclear phagocytes is described in detail by Paul and Balazs in, The mechanism of the regulation of phagocytosis of human polymorphonuclear phagocytes by Na-Hyaluronate. Science (in publication, 1979).
Stossel, in Phagocytosis: Recognition and Ingestion. Seminars in Hematology, Volume 12, 83-116 (1973) ascertained that phagocytosis occurs in two phases: first, the recognition and adhesion and second, the ingestion of internalization. The stimulating effect of Na-HA in low concentrations is due to the enhanced adhesion which in turn increases the rate of ingestion. This stimulation, however, does not cause the cells to ingest more particles than they would without Na-HA. What the stimulation accomplishes is an increase in the total number of particles adhered to the cell surface at any given time. This in turn makes it possible for the rate of ingestion to be increased. That is, the cells complete their maximum level of phagocytosis during a shorter period of time. The inhibition at higher Na-HA concentrations (&gt;100 .mu.g/ml), on the other hand, does not affect the adhesion process at all, but inhibits the ingestion phase of phagocytosis. This effect is entirely dependent on the viscosity of the solution. The viscosity depends on both the concentration and molecular size. Therefore the total inhibition of phagocytosis can be achieved with a 10 mg/ml solution of Na-HA with a MW of 48,000, a 5 mg/ml solution of Na-HA, with a MW of 260,000 or with a 0.4 mg/ml solution of Na-HA with a MW of 3.2 million.
Klockars and Roberts, in Stimulation of phagocytosis by human lysozyme. Acta Haemat. 55, 289-295 (1976) found that muramidase (MUas) stimulates phagocytosis by human polymorphonuclear phagocytes. However, only homologous, i.e., human, but not egg white MUas showed this effect. The stimulation was observed in 10 to 100 .mu.g/ml concentrations of MUas using yeast cells as the phagocyted particles. This concentration of MUas is in the same range as was found in inflammatory exudates by Senn, Chu, O'Malley and Holland, Experimental and Clinical Studies on Murmaidase (Lysozyme). Acta haemat. 44, 65-77 (1970), and in arthritic synovial fluids by Pruzanski, Ogryzlo and Katz, Lysozyme production and abnormalities in rheumatic diseases. In Lysozyme. Academic Press, New York. Chapter 38, 419-425 (1974). Klockars and Roberts, supra, suggested that MUas released by the phagocytes during the ingestion phase (see Wright and Malawista, [The mobilization and extracellular release of granular enzymes from human leukocytes during phagocytosis. J. of Cell Biology, 53, 788-798 (1972)] has an "auto-stimulatory" effect on the cells. MUas, however, is not an opsonin, because it acts on the cell membrane rather than on the particle.